In this reporting period we have made significant progress in our studies. 1. Orai1 and TRPC1 generate distinct local and global Ca2+i signals that underlie their functional specificity. We hypothesized that channels such as Orai1 and TRPC1 generate distinct Ca2+i signals, the spatio-temporal pattern of which determines their functional specificity. To assess this, we have examined the contributions of Orai1 and TRPC1 to carbachol (CCh)-induced Ca2+i signals and Ca2+ dependent gene expression in single cells. Herein we report that endogenous Orai1 and TRPC1 channels contribute to distinct local and global Ca2+i signals following agonist stimulation. In HSG cells, Ca2+ entry via Orai1 controls the generation of Ca2+i oscillations at all CCh tested. In contrast, TRPC1 mediates sustained Ca2+i elevation at relatively high agonist and contributes to the frequency of baseline Ca2+i oscillations at lower agonist. Importantly, the channels display functional specificity in the regulation of Ca2+-dependent transcription factors and gene expression. Consistent with the oscillatory Ca2+i signals generated by Orai1, NFAT translocation and NFAT-dependent gene expression are exclusively dependent on Orai1-mediated Ca2+ entry, without any contribution of TRPC1. Our data suggest that NFAT is regulated by Ca2+i achieved locally near the Orai1 channel, likely due to localization of calmodulin-calcineurin-NFAT within the Orai1-associated microdomain, such that the Ca2+ entering via Orai1 can be locally sensed by the calcium sensor. The exact Ca2+ sensors that decode the two individual Ca2+ signals and the exact amplitude of the signals remain to be determined. 2. Physiological relevance of caveolin 1 in salivary gland fluid secretion. We showed earlier that caveolin1 (Cav1) interacts with TRPC1, serving as a scaffold for the channel within specific PM microdomains and facilitates TRPC1-STIM1 interaction. We have now examined the salivary gland fluid secretion and acinar cell function in Cav1-/- mice. These mice display significant loss of saliva flow as well as CCh- and Tg-stimulated Ca2+ influx. While agonist-induced STIM1-Orai1 interaction is not unaltered, localization of TRPC1, its association with lipid raft domains, and interaction with STIM1 are severely attenuated. In addition, targeting of AQP5 to the apical region of acinar cells is also impaired. Preliminary observations also indicate defects in trafficking of secretory granules. Finally, adenovirus-mediated in vivo expression of Cav1 in SMG of Cav1-/- mice induces recovery of agonist-stimulated TRPC1-STIM1 association, store-dependent Ca2+ entry, AQP5 localization and fluid-secretion. Together, these findings suggest that Cav1 has a critical role in the trafficking of key proteins in the salivary gland, including TRPC1 and AQP5. 3. STIM1 and STIM2 deficiency in T lymphocytes underlies development of exocrine gland autoimmune disease, Sjgrens Syndrome. In contrast to loss of Orai1, which causes severe combined immune deficiency (SCID), patients with mutations in STIM1 display autoimmunity and lymphoproliferation. Primary Sjgrens syndrome (pSS) is a chronic autoimmune disease affecting exocrine glands, primarily salivary and lacrimal glands, resulting in gland destruction, xerostomia (dry mouth) and keratoconjunctivitis sicca (dry eyes). There are no conclusive data that establish the molecular basis for the disease, either in the salivary gland itself or systemically in cells such as lymphocytes. We used a mouse model with T lymphocyte targeted deletion of STIM1 and STIM2 which displayed signs of autoimmunity including decreased Treg, dermatitis, blepharitis, and infiltration of lymphocytes into epithelial tissues such as liver and lungs to examine a possible link between STIM1 and STIM2 deficiency in T cells and autoimmunopathy of the salivary glands. We report here that mice with T-cell-targeted deletion of STIM and STIM2 develop spontaneous and severe pSS-like autoimmune disease. Diffuse lymphocytic infiltration was seen in submandibular glands, a major target of pSS, by 6 weeks progressing to severe inflammation by 12 weeks of age in the knockout mice. pSS-specific autoantibodies (SSA/Ro and SSB/La) were detected in the serum, with progressive salivary gland destruction and loss of fluid secretion. Importantly, PMBCs as well as lymphocytic infiltrates in submandibular glands from pSS patients demonstrated significant reduction in STIM1 and STIM2 proteins as a function of the level of inflammation. Store-operated calcium entry was also reduced in PBMCs from pSS patients compared to those from healthy individuals. Together our findings reveal a novel link between STIM1 and STIM2 proteins and pSS. The exact mechanism by which STIM protein levels are altered in the lymphocytes is not clear. Our studies indicate that STIM1 levels in glands from pSS patients are also reduced. Ongoing studies in the laboratory (in collaboration with Ilias Alevizos, SS Clinic MPTB) have identified several miRs that are elevated in pSS patients that can potentially target STIM1. We believe that within the salivary gland milieu cytokines, miRs, or other epigenetic factors, can alter gene expression and/or cell function. 4. Irradiation leads to activation of TRPM2: radiation-induced irreversible salivary gland dysfunction is prevented by deletion of TRPM2 or suppression of channel activation. Debilitating side effects of IR in head and neck cancer patients include oral mucositis and xerostomia, which continue to exist post therapy and are primarily due to persistent salivary gland hypofunction. There are no adequate therapies to protect against, or reverse, IR-induced salivary hypofunction. We have directed our studies to identify new targets for IR. Transient Receptor Potential Melastatin-like 2 (TRPM2) has been implicated in the amplification of ROS-induced signaling, cell damage, and chronic inflammation. We report for the first time that functional TRPM2 channel is present in salivary gland cells and is activated in reponse to IR. Importantly, loss of TRPM2, or suppression of function, protects against IR-induced irreversible salivary gland dysfunction. Together, our findings provide evidence that TRPM2 contributes to IR-induced irreversible salivary gland dysfunction and suggest that this channel can be proposed as a novel target for attenuating the effects of IR on healthy salivary gland tissues. 5. Sialin (SLC17A5) functions as a nitrate transporter in the plasma membrane. More than 25% of the circulating NO3- is actively removed and secreted by salivary glands where oral commensal bacteria convert it to NO2-, which enters circulation and gets converted to NO. Thus, salivary NO3- provides a non-canonical pathway for regulating serum NO homeostasis. The transport mechanisms involved in NO3- uptake and secretion have not yet been identified. We have now identified that sialin (SLC17A5), mutations in which cause Salla disease and infantile sialic acid storage disorder (ISSD), functions as an electrogenic 2NO3-/H+ cotransporter in the plasma membrane of salivary gland acinar cells involved in NO3- uptake into the gland. Knockdown of sialin expression decreased while a plasma membrane-targeted mutant (L22A-L23A) increased NO3- uptake into salivary gland cells. Fibroblasts from ISSD patients displayed reduced SA- and NO3--currents compared to healthy controls while expression of disease-associated sialin mutants in healthy fibroblasts and salivary gland cells suppressed NO3- conductance. Importantly, adenovirus-dependent expression of the sialinH183R mutant in vivo in pig salivary glands decreased NO3- secretion in saliva. We suggest that sialin can contribute significantly to clearance of serum nitrate by salivary glands.